Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Pre-conditioning induced by carbon monoxide provides neuronal protection against apoptosis

Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme-oxygenase, presenting anti-apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme-oxygenase product critical for its anti-apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre-conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP-dependent mitochondrial K channel (mitoK(ATP)) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoK(ATP) abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoK(ATP), which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies.     

Paracrine neuroprotective effect of nitric oxide in the developing retina

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.     

Association of the tyrosine phosphatase SHP-2 with transducin-alpha and a 97-kDa tyrosine-phosphorylated protein in photoreceptor rod outer segments

Increasing evidence indicates that tyrosine phosphorylation, controlled by the concerted action of tyrosine kinases and protein tyrosine phosphatases (PTPs), plays important roles in retinal photoreceptor rod outer segments (ROS). We characterized PTP activity in isolated bovine ROS that is significantly inhibited by orthovanadate. Incubating ROS in the presence of exogenous Mg2+, ATP, and orthovanadate dramatically enhanced the tyrosine phosphorylation of several endogenous proteins. SHP-2, a PTP with two SH2 domains, was identified in ROS by immunoblot analysis and was found to associate with ROS membranes. Immunocytochemistry showed localization of SHP-2 in photoreceptor outer segments and possibly in the outer plexiform, inner nuclear, and inner plexiform cell layers of the retina as well. SHP-2 associated with transducin-alpha and a 97-kDa tyrosine-phosphorylated protein in ROS, suggesting the formation of a multimeric signaling complex. Based on its association with transducin-alpha and a 97-kDa protein, SHP-2 may regulate the tyrosine phosphorylation of endogenous proteins, including transducin-alpha, and may play a significant role in a novel signaling pathway in photoreceptors.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.     

Localization of actin and tubulin in developing and adult mammalian photoreceptors

Summary In order to define cytoskeletal domains of the mammalian photoreceptor, actin and tubulin were localized in adult retinae of mouse and human. For light-microscopic localization, actin was labeled using fluorescent phalloidin or monoclonal antibodies against actin, and tubulin was labeled using monoclonal antibodies against alpha- and beta-tubulin in an immunocytochemical method. Actin and tubulin were also localized by ultrastructural immunocytochemistry in the mouse. Filamentous actin was present in the retina at the outer limiting membrane and in synaptic terminals, especially of the cones, while globular actin was observed additionally in the inner segments. Müller cell cytoplasm and apical microvilli at the outer limiting membrane were also labeled for filamentous actin. Alpha- and beta-tubulin were evident throughout the photoreceptors, including the inner segments, but not in the synaptic terminals or at the outer limiting membrane. In the early postnatal retina of mouse, actin and tubulin were present at the ventricular surface. This pattern changed as photoreceptors fully elongated and as synaptogenesis occurred in the outer plexiform layer.     

Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.     

Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca(2+) channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba(2+) as the charge carrier. Maximal peak Ba(2+) current (I(Ba)) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 +/- 3%). The NO donor S-nitroso-N-acetylpenicillamine also increased I(Ba), and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in I(Ba) was blocked when cells were pretreated with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 microM) and milrinone (3 microM), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on I(Ba). Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca(2+) channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO.     

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.     

Altered regulation of nitric oxide and natriuretic peptide system in cisplatin-induced nephropathy

Cisplatin is a chemotherapeutic agent used for treating solid tumors. However, nephrotoxicity is the dose-limiting factor in its clinical use. The present study was aimed to determine whether altered regulation of the local nitric oxide (NO) and natriuretic peptide (NP) systems is involved in the pathogenesis of cisplatin-induced nephropathy. Cisplatin (6 mg/kg) was injected intraperitoneally into male Sprague-Dawley rats. The control group was not treated with cisplatin. Expression levels of nitric oxide synthase (NOS), nitrotyrosine, soluble guanylyl cyclase and neutral endopeptidase (NEP) in the kidneys were determined 4 days after treatment by semiquantitative immunoblotting. mRNA expression of NPs and natriuretic peptide receptors (NPRs) was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclase were determined by measuring the amount of cyclic 3',5'-guanosine monophosphate (cGMP) generated in responses to sodium nitroprusside and atrial natriuretic peptide (ANP), respectively. In the test rats, creatinine clearance was decreased, while sodium and water excretion were increased. The expression of inducible NOS (iNOS) and nitrotyrosine was increased in the cortex/outer stripe of outer medullar and inner medullar, while that of endothelial and neuronal NOS was decreased in the inner medullar. Excretion of NO metabolites was increased in these rats. The catalytic activity of soluble guanyly cyclase was blunted in the papilla after cisplatin was administered. The mRNA expression of ANP, brain natriuretic peptide, and C-type natriuretic peptide was increased, while that of NPR-A and NPR-C were decreased in the test rats. The catalytic activity of soluble and particulate guanylyl cyclase in the papilla was blunted after cisplatin was administered. In conclusion, increased production of NO by iNOS may contribute to cytotoxic injury, resulting in cisplatin-induced nephropathy, while the up-regulation of renal natriuretic peptide synthesis together with the down-regulation of NEP and NPR-C may contribute to the natriuresis and diuresis seen in cisplatin-induced nephropathy.     

Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.